The recent expansion of the world market for High Fructose Corn Syrup (HFCS) makes potential improvements in HFCS production that would result from increased isomerization yields, increased cost effectiveness and elimination of non-enzymatic browning (Maillard browning reaction) by-products considerable. HFCS producing industries are interested in a highly stable xylose (glucose) isomerase that is active at about 60° C. and slightly acidic pH instead of at neutral or slightly basic pH. A thermo-acid-stable xylose (glucose) isomerase would be used to eliminate the onset of by-products of browning reactions and increase the time between biocatalyst replacements.
Xylose isomerase (XI) converts D-xylose to D-xylulose in vivo and also catalyzes the conversion of D-glucose to D-fructose in vitro. This latter activity is used in industry for the production of high fructose corn syrup (HFCS). XI is one of the largest volume commercial enzymes used today. Typically, the pH optima of commercially available glucose isomerases range from about 7.5 to about 9.0. This range limits the reaction temperature used in the industrial glucose isomerization process to around 60° C. due to the formation of browning products by non-enzymatic reactions between reducing sugars and proteins at higher temperatures and alkaline pHs. Thermostable XIs with neutral or slightly acidic pH optima have a potential for industrial applications with the advantages of faster reaction rates, higher fructose concentrations at equilibrium, higher process stability, decreased viscosity of substrate and product streams, and fewer problems with by-product formation.
Numerous and intensive site-directed mutagenesis studies have been performed to improve enzyme catalysts. Despite these efforts, no generally applicable rules have been established to develop acid-stable or thermostable enzyme catalysts. Besides, rules for engineering protein stability and activity by rational design are likely to be protein-specific, and any such design effort would need to be guided by detailed structural information. Directed evolution, on the other hand, has proved to be useful for modifying enzymes in the absence of such knowledge.
An ideal XI suitable for use in the HFCS industry should meet the following conditions: (a) it should function under acidic conditions (pH of about 6 or less) to avoid browning reactions; (b) it should function under higher reaction temperatures than current commercial XIs and lower reaction temperatures than thermophilic xylose isomerases' optimal temperature for activity, that is, from about 60° C. to about 80° C.; and (c) it should retain high stability at high temperature.
It would require enormous effort, time, and structural knowledge to use a site-directed mutagenesis approach to try to engineer such an enzyme. Directed evolution has proved to be useful for modifying enzymes in the absence of structural knowledge (Kuchner and Arnold, 1997). In directed evolution, the process of natural evolution is accelerated in a test tube for selecting proteins with the desired properties (Moore and Maranas, 2000).
A basic protocol for directed evolution starts with the creation of a library of mutated genes encoding for the protein of interest, usually by means of error-prone PCR (also known as random mutagenesis). Once created, these genes are then ligated into an expression vector and transformed into suitable bacterial cells. A screening procedure is then applied to isolate the few transformants containing mutated genes that encode for proteins with improved properties. Proteins with different properties can be recombined using a DNA shuffling approach (Stemmer, 1994 and Zhao and Arnold, 1997).
U.S. Pat. Nos. 5,219,751, 5,268,280, 5,656,497, and 5,935,837, which are hereby incorporated by reference in their entirety, disclose glucose isomerases obtained from Thermotoga maritima and Thermotoga neapolitana. However, these patents do not disclose DNA constructs encoding enzymes that are capable of preparing D-fructose by enzymatically treating D-glucose with the xylose isomerase at a lower reaction temperature (from about 45° C. to about 100° C.) and under acidic conditions (pH of about 5.2 to about 7.0) to obtain a syrup containing up to about 60–65% D-fructose.
Vieille et al., Appl. Environ. Microbiol. 1995 61 (5) 1867–1875, describe a gene derived from a strain of Thermotoga neapolitana, encoding a xylose isomerase, which consists of 444 amino acid residues, and has a calculated molecular weight of 50,892.
Previous attempts have been made to obtain thermostable xylose isomerases, either by site-directed mutagenesis of moderately thermostable xylose isomerases, or by screening highly thermophilic organisms for xylose isomerase activity. However, none of those attempts have resulted in commercially useful xylose isomerases that allow processing of sugars at a reduced temperature.
Therefore, it would be advantageous to have a xylose isomerase with enhanced stability, activity, and utility and an efficient method of producing that enzyme in quantity.
Objects
It is an object of the present invention to disclose a novel xylose isomerase (“XI”).
It is a further object to disclose a method of producing the enzyme employing DNA encoding for the enzymes, plasmids containing the DNA, and bacteria into which the plasmids have been inserted and which produce the enzyme.
It is a still further object to disclose a method of making fructose using the novel xylose isomerase.
The present invention relates to a xylose isomerase, which is obtained from the microorganism Thermotoga neapolitana and nucleic acids encoding for such isomerase. The enzyme has the amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:11. Preferably, the enzyme has the amino acid sequence SEQ ID NO:4, which is:
TNX1 3A2 (V185T,L282P)MAEFFPEIPK VQFEGKESTN PLAFKFYDPE EIIDGKPLKD HLKFSVAFWH TFVNEGRDPF60 GDPTADRPWN RYTDPMDKAF ARVDALFEFC EKLNIEYFCF HDRDIAPEGK TLRETNKILD120 KVVERIKERM KDSNVKLLWG TANLFSHPRY MHGAATTCSA DVFAYAAAQV KKALEITKEL180 GGEGYTFWGG REGYETLLNT DLGFELENLA RFLRMAVDYA KRIGFTGQFL IEPKPKEPTK240 HQYDFDVATA YAFLKSHGLD EYFKFNIEAN HATLAGHTFQ HEPRMARILG KLGSIDANQG300 DLLLGWDTDQ FPTNVYDTTL AMYEVIKAGG FTKGGLNFDA KVRRASYKVE DLFTGHIAGM360 DTFALGFKVA YKLVKDGVLD KEIEEKYRSF REGIGRDIVE GKVDFEKLEE YIIDKETIEL420 PSGKQEYLES LINSYIVKTI LELR444
The preferred enzyme has the amino acid sequence SEQ ID NO:11, which is:
TNXI 1F1 (V185T/F186S/L282P)deduced amino acid sequence 1        .         .         .         .         .          .         .         80AEFFPEIPKVQFEGKESTNPLAFKFYDPEEIIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTADRPWNRYTDPMDKAFA 81       .         .         .         .         .         .         .         160RVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSAD 161      .         .         .         .         .         .         .         240VFAYAAAQVKKALEITKELGGEGYTSWGGREGYETLLNTDLGFELENLARFLRMAVDYAKRIGFTGQFLIEPKPKEPTKH 241      .         .         .         .         .         .         .         320QYDFDVATAYAFLKSHGLDEYFKFNIEANHATLAGHTFQHEPRMARILGKLGSIDANQGDLLLGWDTDQFPTNVYDTTLA 321      .         .         .         .         .         .         .         400MREVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKVAYKLVKDGVLDKFIEEKYRSFREGIGRDIVEG 401      .         .         .         .         .         .         .         480KVDFEKLEEYIIDKETIELPSGKQEYLESLINSYIVKTILELR*******This is TNX1F1 (V185T, F1865, L282P) or SEQ ID NO:11 (FIG. 19).
The preferred method of the present invention for producing the enzyme, comprises, isolating and purifying xylose isomerase gene, partially digesting the DNA with a restriction enzyme, ligating the DNA into a plasmid vector, transforming the E. coli with the ligation mixture, growing the E. coli and isolating the enzyme from the E. coli. 
The novel isolated xylose isomerase gene has the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9. Preferably, the novel isolated xylose isomerase gene has the nucleotide sequence of SEQ ID NO:3. The nucleotide sequence SEQ ID NO:3 is:
TNX1 3A2 (V185T,L282P)gtcgacgcaa aggtcgtgac gggtggaaac ataaacgttc agctgggaac tgtgtcctcg60 gctgctgttg aaggaacata cgttatcgaa gttggacaat tctctggaac ggtcacatcc120 gagcttgatg tcaagatccg ccgttgtcct cagcacccct tccgtacacc ctgtcatcct180 tcacaacggg gatgaaggga tccgtttccc acagcgaaag atcccctggt ggaacggtgt240 ctatgtgtgt cactatccac aatgttttgc ttctgtccct gccgggaatg attgcaagca300 gattcgacct ccaaattccg ttctggtctt ttgtgtcatg acgctcaaca gtgtatccca360 tctttttgag aagttcctcc agccagtcgg ccttctcttt ctctccaggt ccaccgaaga420 ctggattcac cgaattgatc gatatgaacc ttttcagcga atctaccatt tcgtctttca480 attcttctat ctttcttgtt atctccatct gaaacacctc ccaagtacaa gtatatctct540 ccaaaaaaat atttgaaatg accccaggga attttatata attgattgat agaaaaaatt600 tagggaggtg ttcacatggc tgaattcttt ccagaaatcc cgaaagtgca gttcgaaggc660 aaagaaagca caaatccact tgcgttcaag ttctacgatc cagaagagat catcgacggc720 aaacccctca aggaccatct gaagttctcc gttgccttct ggcacacctt cgtgaacgag780 gaagggatc ccttcggaga cccaacggcc gatcgtccct ggaacaggta caccgatccc840 atggacaagg cttttgcaag ggtggacgcc ctttttgaat tctgcgaaaa actcaacatc900 gagtacttct gcttccacga cagagacatc gctcccgagg gaaaaacgct gagggagaca960 aacaaaattt tggacaaagt agtggagaga atcaaagaga gaatgaaaga cagcaacgtg1020 aagctcctct ggggtactgc aaacctcttt tcccacccaa ggtacatgca tggtgcagcg1080 acaacctgca gtgctgatgt ttttgcgtac gcggccgccc aggtgaaaaa agcccttgag1140 atcaccaaag aacttggagg agaagggtac accttctggg gtggaagaga aggatacgaa1200 acactcctca acacggacct tggattcgaa cttgaaaacc tcgcccgctt cctcagaatg1260 gctgtggatt atgcaaaaag gatcggtttc accggacagt tcctcatcga accaaaaccg1320 aaagaaccca ccaaacacca gtacgacttc gacgttgcaa ccgcctatgc cttcctgaag1380 agccacggtc tcgatgaata cttcaaattc aacatcgagg caaaccacgc cacactcgcc1440 ggtcacacct tccagcacga accgagaatg gcaaggatcc ttggaaaact cggaagcatc1500 gatgcaaacc agggagacct tcttcttgga tgggacaccg atcagttccc aacaaacgtc1560 tacgatacaa cccttgcaat gtacgaagtg ataaaagcgg gaggcttcac aaaaggtggg1620 ctcaacttcg atgcgaaggt gaggagggct tcttacaaag tggaggacct cttcataggg1680 cacatagcgg gaatggacac ctttgcactc ggtttcaagg tggcatacaa actcgtgaag1740 gatggtgttc tggacaaatt catcgaagaa aagtacagaa gtttcaggga gggcattgga1800 agggacatcg tcgaaggtaa agtggatttt gaaaaacttg aagagtatat aatagacaaa1860 gaaacgatag aacttccatc tggaaagcaa gaatacctgg aaagcctcat caacagttac1920 atagtgaaga ccattctgga actgaggtga aacagagtgt gaagttcttg aatcttcgaa1980 gattacttct tctggcactg attgcggctg gaatctcagt gatcatagtc gtatccaacc2040 gggaaaacag ggtgaaattt ccagaaggag agattgtgat aactgacgga gaaagatctc2100 tgaaacttcg tgtcgagata gcgaacactc ctttttttcg ttcgatcggt ctgatgtaca2160 gaaagagcat cccggatgac ttcgggatgc tctttgtttt tgaagaagat acaagaagcg2220 gcttctggat gaagaacacc tacgttcccc tcgaaatcgc cttcatagac agaaacggca2280 tcgtattttc cattcaggag atggagccat gcgaaaaaga accctgcaag gtttactacg2340 caccaaagcc gttcagatac gctcttgaag tgaaaagagg ttttttcgaa aggcatggat2400 ttggagtggg aagccgtgtc ctgatagaaa agtagcggta ctttcaaaca aaaacgtatg2460 gaatcttcat cttctttgcc tcgtacattc tcgagtcagc catcttcaga agttcttcta2520
The nucleotide sequence of SEQ ID NO:12 is:
TNXI 1F1 nucleotide sequence (ORF starting at 616 bp)1        .         .         .         .         .         .         .         80GTCGACGCAAAGGTCGTGACGGGTGGAAACATAAACGTTCAGCTGGGAACTGTGTCCTCGGCTGCTGTTGAAGGAACATA 81       .         .         .         .         .         .         .         160CGTTATCGPAGTTGGACAATTCTCTGGAACGGTCACATCCGAGCTTGATGTCAAGATCCGCCGTTGTCCTCAGCACCCCT 161      .         .         .         .         .         .         .         240TCCGTACACCCTGTCATCCTTCACAACGGGGATGAGGGATCCGTTTCCCACAGCGAAGATCCCCTGGTGGAACGGTGT 241      .         .         .         .         .         .         .         320CTATGTGTGTCACTATCCACAATGTTTTGCTTCTGTCCCTGCCGGGAATGATTGCAAGCAGATTCGACCTCCAAATTCCG 321      .         .         .         .         .         .         .         400TTCTGGTTTTTGTGTCATGACGCTCAACAGTGTATCCCATCTTTTTGAGAAGTTCCTCCAGCCAGTCGGCCTTCTCTTT 401      .         .         .         .         .         .         .         480CTCTCCAGGTCCACCGAAGACTGGATTCACCGAATTGATCGATATGAACCTTTTCAGCGAATCTACCATTTCGTCTTTCA 481      .         .         .         .         .         .         .         560ATTCTTCTATCTTTCTTGTTATCTCCATCTGAAACACCTCCCAAGTACAAGTATATCTCTCCAAAAAAATATTTGAAATG 561      .         .         .         .         .       ***  .         .         640ACCCCAGGGAATTTTATATAATTGATTGATAGAAAAAATTTAGGGAGGTGTTCACATGGCTGAATTCTTTCCAGAAATCC                                                           A  E  F  F  P  E  I 641      .         .         .         .         .         .         .         720CGAAAGTGCAGTTCGAAGGCAAAGAAAGCACAAATCCACTTGCGTTCAAGTTCTACGATCCAGAAGAGATCATCGACGGCP  K  V  Q  K  E  G  K  E  S  T  N  P  L  A  F  K  F  Y  D  P  E  E  I  I  D  G 721      .         .         .         .         .         .         .         800AAACCCCTCAAGGACCATCTGAAGTTCTCCGTTGCCTTCTGGCACACCTTCGTGAACGAGGGAAGGGATCCCTTCGGAGA K  P  L  K  D  H  L  K  F  S  V  A  F  W  H  T  F  V  N  H  G  R  D  P  F  G  D 801      .         .         .         .         .         .         .         880CCCAACGGCCGATCGTCCCTGGAACAGGTACACCGATCCCATGGACAAGGCTTTTGCAAGGGTGGACGCCGTTTTTGAAT  P  T  A  D  R  P  W  N  R  Y  T  D  P  M  D  K  A  F  A  R  V  D  A  L  F  E 881      .         .         .         .         .         .         .         960TCTGCGAAAAACTCAACATCGAGTACTTCTGCTTCCPCGACAGAGACATCGCTCCCGAGGGAAAAACGCTGAGGGAGACAF  C  E  K  L  N  I  E  Y  F  C  F  H  D  R  D  I  A  P  H  G  K  T  L  R  E  T 961      .         .         .         .         .         .         .         1040AACAAAATTTTGGACAAAGTAGTGGAGAGAATCAAAGAGAGAATGAAAGACAGCAAGGTGAAGCTCCTCTGGGGTACTGC N  K  I  L  D  K  V  V  E  R  I  K  E  R  M  K  D  S  N  V  K  L  L  W  G  T  A 1041     .         .         .         .         .         .         .         1120AAACCTCTTTTCCCACCCAAGGTACATGCATGGTGCAGCGACAACCTGCAGTGCTGATGTTTTTGCGTACGCGGCCGCCC  N  L  F  S  H  P  R  Y  M  H  G  A  A  T  T  C  S  A  D  V  F  A  Y  A  A  A 1121     .         .         .         .         .         .         .         1200AGGTGAAAAGCCCTTGAGATCACCAAGAACTTGGAGGAGAAGGGTACACCTCCTGGGGTGGAAGAGAAGGATACGAAQ  V  K  K  A  L  E  I  T  K  E  L  G  G  E  G  Y  T  S  W  G  G  R  E  G  Y  E 1201     .         .         .         .         .         .         .         1280ACACTCCTCAACACGGACCTTGGATTCGAACTTGAAAACCTCGCCCGCTTCCTCAGAATGGCTGTGGATTATGCAAAAAG T  L  L  N  T  O  L  G  F  E  L  H  N  L  A  R  F  L  R  M  A  V  D  Y  A  K  R 1281     .         .         .         .         .         .         .         1360GATCGGTTTCACCGGACAGTTCCTATCGAACCAAAACCGAAAGAACCCACCAACACCAGTACGACTTCGACGTTGCAA  I  G  F  T  G  Q  F  L  I  H  P  K  P  K  E  P  T  K  H  Q  Y  O  F  D  V  A 1361     .         .         .         .         .         .         .         1440CCGCCTATGCCTTCCTGAAGAGCCACGGTCTCGATGAATACTTCAAATTCAACATCGAGGCAACCACGCCACACTCGCC 1441     .         .         .         .         .         .         .         1520GGTCACACCTTCCAGCACGAACCGAGAATGGCAAGGATCCTTGGAAAACTGGAAGCATCGATGCAAACCAGGGAGACCT G  H  T  F  Q  H  E  P  R  M  A  R  I  L  G  K  L  G  S  I  D  A  N  Q  G  D L 1521     .         .         .         .         .         .         .         1600TCTTCTTGGATGGGACACCGATCAGTTCCCAACAAACGTCTACGATACAACCCTTGCAATGTACGAAGTGATAAAAGCGG  L  L  G  W  D  T  D  Q  F  P  T  N  V  Y  D  T  T  L  A  M  Y  E  V  I  K  A 1601     .         .         .         .         .         .         .        1680GAGGCTTCACAAGGTGGGTCAACTTCGATGGGAAGGTGAGGAGGGCTTCTTACAAAGTGGAGGACCTCTTCATAGGGG  G  F  T  K  G  G  L  H  F  D  A  K  V  R  R  A  S  Y  K  V  E  D  L  F  I  G 1681     .         .         .         .         .         .         .        1760CACTAGCGGGAATGGACACCTTTGCACTCGGTTTCAAGGTGGCATACACTCGTGAAGGATGGTGTTCTGGACAAATT H  I  A  G  M  D  T  F  A  L  G  F  K  V  A  Y  K  L  V  K  D  G  V  L  D  K  F 1761     .         .         .         .         .         .         .         1840CATCGAAGAAAAGTACAGAAGTTCAGGGAGGGCATTGGAAGGGACATCGTCGAAGGTAAAGTGGATTTTGAAAAACTTG  I  E  E  K  Y  R  S F  R  E  G  I  G  R  D  I  V  E  G  K  V  D  F  E  K  L 1841     .         .         .         .         .         .         .        1920AAGAGTATATAATAGACAAGAAACGATAGAACTTCCATCTGGAAAGCAAGAATACCTGGAAAGCCTCATCAACAGTTACE  E  Y  I  I  D  K  E  T  I  E  L  P  S  G  K  Q  E  Y  L  E  S  L  I  H  S  Y 1921     .         .       ***         .        .         .         .         2000ATAGTGAAGACCATTCGGAACTGAGGTGAAACAGAGTGTGAAGTTCTTGAATCTTCGAAGATTACTTCTTCTGGCACTG I  V  K  T  I  L  E  L R ATTGCGGCTGGAATCTCAGTGATCATAGTCGTATCCAACCGGGAAAACAGGGTGAAATTTCCAGAAGGAGAGATTGTGATAACTGACGGAGAAAGATCTCTGAAACTTCGTGTCGAGATAGCGAACACTCCTTTTTTTCGTTCGATCGGTCTGATGTACAGAAAGAGCATCCCGGATGACTTCGGGATGCTCTTTGTTTTTGAAGAAGATACAAGAAGCGGCTTCTGGATGAAGAACACCTACGTTCCCCTCGAAATCGCCTTCATAGACAGAAACGGCATCGTATTTTCCATTCAGGAGATGGAGCCATGCGAAAAAGAACCCTGCAAGGTTTACTACGCACCAAAGCCGTTCAGATACGCTCTTGAAGTGAAAAGAGGTTTTTTCGAAAGGCATGGATTTGGAGTGGGAAGCCGTGTCCTGATAGAAAAGTAGCGGTACTTTCAAACAAAAACGTATGGAATCTTCATCTTCTTTGCCTCGTACATTCTCGAGTCAGCCATCTTCAGAAGTTCTTCTAGA TNXI 1F1 [V185T, F186S, and L282P]185:GTC(Val)→ACC(Thr)186:TTC (Phe)→TCC (Ser)282:CTG (Leu)→CCG (Pro)The novel recombinant plasmid comprises a compatible vector containing the DNA sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 or SEQ ID NO:12. A compatible vector is one into which the gene can be inserted and which can be introduced into a suitable host for production of the enzyme.
The preferred method of preparing D-fructose comprises enzymatically treating D-glucose with the xylose isomerase of the present invention at a temperature of about 45° C. to about 100° C. at a pH of about 5.2 to about 8.0 to obtain a syrup containing up to about 50 to about 60% D-fructose.
The achievement of the above and other objects and advantages of the present invention will be apparent to those skilled in the art from the description of the drawings, the preferred embodiment and the experimental work.
In one aspect, the invention provides a purified polypeptide that includes an amino acid sequence at least 80% identical to amino acids 2–444 of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. A preferred polypeptide is an amino acid sequence at least 95% identical to amino acids 2–444 of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:11.
In another aspect, the invention provides an isolated nucleic acid molecule encoding an XI polypeptide. In preferred embodiments, the encoded polypeptide comprising an amino acid sequence at least 80% identical to amino acids 2–444 of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12.
A preferred nucleic acid encodes a polypeptide that includes an amino acid sequence at least 95% identical to amino acids 2–444 of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:11.
The invention also includes an oligonucleotide that includes a portion of an XI encoding nucleic acid. For example, the oligonucleotide can be at least 10 nucleotides in length and include at least nine contiguous nucleotides of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:12.
Also provided by the invention is a vector that includes an XI encoding nucleic acid. The vector can include, e.g., a nucleic acid encoding an XI polypeptide that includes an amino acid sequence at least 80% identical to amino acids 2–444 of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:11. In a further aspect, the invention includes a cell that includes the XI nucleic acid-containing vector.
In a further aspect, the invention provides an antibody that selectively binds to an XI polypeptide, e.g., a polypeptide that includes an amino acid sequence at least 80% identical to amino acids 2–444 of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:11. The antibody can be a polyclonal antibody or a monoclonal antibody. In some embodiments, the antibody neutralizes the isomerase activity of an XI polypeptide.
The invention also includes a method of producing an XI polypeptide by culturing a cell that includes an XI-encoding nucleic acid under conditions allowing for expression of the polypeptide encoded by the XI nucleic acid.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.